Methods for synthesizing polymers (e.g., oligonucleotides) on a solid support have been developed for producing large arrays of polymer sequences on solid substrates. These large “arrays” of polymer sequences have wide ranging applications and are of substantial importance to many industries including, but not limited to, the pharmaceutical, biotechnology and medical industries. For example, the arrays can be used in screening large numbers of molecules for biological activity, e.g., receptor binding capability. And arrays of oligonucleotide probes can be used to identify mutations in known sequences, as well as in methods for de novo sequencing of target nucleic acids. In addition, PNA (peptide nucleic acids) arrays can be used to screen molecules which are useful in antisense (mRNA) gene regulation, or molecules which bind to specific sequences of double-stranded DNA. Furthermore, combinatorial arrays can be used in gene expression analysis. See, for example, van Dam et al., Genome Research, 2002, 12, 145–152, which is incorporated herein by reference in its entirety.
Oligonucleotide arrays with up to hundreds of thousands of samples on an area of a few square centimeters have been synthesized and proven to be extraordinarily useful in various applications including gene expression studies. Over the past several years, a new set of technologies have emerged for making arrays of synthetic surface-bound polymers.
One method for synthesizing high-density patterns on surfaces is photolithography process as discussed in U.S. Pat. No. 5,143,854, issued to Pirrung et al., and PCT Application No. 92/10092. In this method, light is directed to selected regions of a substrate to remove protecting groups from the selected regions of the substrate. Thereafter, selected molecules are coupled to the substrate, followed by additional irradiation and coupling steps. By activating selected regions of the substrate and coupling selected monomers in precise order, one can synthesize an array of molecules having any number of different sequences, where each different sequence is in a distinct, known location on the surface of the substrate. This method requires specialized reagents (e.g., photoremovable protecting groups), which is relatively expensive and presently have significantly lower coupling yield than conventional reagents. Moreover, in general, to make an array of N-mers requires 4N cycles of deprotection and coupling, one for each of the 4 bases, times N base positions. This photolithographic method also typically requires 4N masks, thereby adding a considerable expense to the procedure. Furthermore, any decreased deprotection efficiency results in the decreased coupling efficiency. And because the deprotection reaction generally does not result in 100% cleavage of the protecting groups, there can be “deletion sequences.” For example, if one strand is accidentally not deprotected, but becomes deprotected later, then it will have missed one or more coupling steps. Conventional reagents do not suffer this problem because it is the coupling step which is most inefficient, and missed couplings can be terminated (nearly completely) with the capping step. Thus with conventional reagents, impurities consist of truncated sequences, but with photo-cleavable reagents, impurities consist of truncated and deletion sequences.
Another method for producing polymer arrays is an ink-jet technique which uses the print heads of commercial piezoelectric ink-jet printers to deliver reagents to individual spots on the array. While this technique uses relatively inexpensive conventional chemical reagents with typically high coupling yield, it can deliver one, and only one, drop of reagents at a time, unless multiple jets are used simultaneously. Moreover, the solid support must be patterned to achieve small feature sizes. Furthermore, two drops of liquid applied too closely together on a surface tend to spread into each other and mix, thereby limiting the array density achievable with the ink-jet method.
There are other methods including robotic deposition of reagents in an array of fluid-containing wells and the use of fluidics to deposit reagents on a surface. See for example, U.S. Pat. No. 6,001,311, issued to Brennan et al. and U.S. Pat. No. 6,121,048, issued to Zaffaroni et al. However, each method has its own limitations such as limited array density, increased production cost per array, and/or serial (i.e., non-parallel) synthesis.
Therefore, there is a need for a chemical reaction apparatus and a method for preparing array of compounds with high throughput, high product quality, enhanced miniaturization and lower costs.